?DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify novel G protein-coupled receptor (GPCR) cytoprotective signaling pathways that can restore or enhance endothelial barrier integrity to prevent vascular leakage associated with sepsis. GPCRs represent the largest family of signaling receptors expressed in mammalian cells and the largest class of drug targets for approved therapeutics. Two GPCRs have been shown to mediate endothelial cytoprotective responses, protease-activated receptor-1 (PAR1) and the sphingosine 1-phosphate receptor-1 (S1PR1). PAR1 is a GPCR for thrombin, but can be cleaved and activated by activated Protein C (APC). APC bound to its co-factor EPCR cleaves the N-terminus of PAR1 at a unique site generating a distinct tethered ligand that promotes cytoprotective signaling. S1PR1 signaling also promotes endothelial barrier maintenance and anti-apoptotic responses and contributes to APC/PAR1-induced cytoprotection. Thus, both PAR1 and S1PR1 make important contributions to endothelial cytoprotection. However, the mechanism by which APC/PAR1 and S1PR1 coordinate cytoprotective signaling in vitro and in vivo is not known and critical to understand to advance the status of these receptors as drug targets for the development of new therapeutics for the prevention and treatment of sepsis. We hypothesize that b-arrestins coordinate APC/PAR1 and S1/S1PR1 signaling in caveolae to promote endothelial cytoprotective responses. We found that APC/PAR1- induced endothelial cytoprotection requires PAR1 localization in caveolae. In recent work, we discovered that APC/PAR1-promoted cytoprotective signaling is mediated by -arrestin-2 and dishevelled-2 scaffolds rather than by heterotrimeric G proteins. In preliminary studies we show that PAR1 and S1PR1 co-associate at the plasma membrane and co-exist in caveolae. Moreover, APC/PAR1 induces activation of sphingosine kinase-1 (SK1), an important mediator of S1P generation and S1PR1 activation, through a -arrestin-2-dependent pathway. -arrestins are also critical for APC-induced anti-apoptotic responses. We further demonstrate using zebrafish that PAR1, S1PR1 and -arrestin-2 regulate vascular permeability in a sepsis model. We will pursue a multidisciplinary approach to identify endothelial GPCR cytoprotective protective signaling pathways in vitro using cultured human endothelial cells and in vivo using a zebrafish model of vascular permeability and sepsis. The proposed studies will advance our understanding of how PAR1 and S1PR1 function to regulate vascular endothelial barrier integrity and apoptosis normally and in a sepsis model. The specific aims of the proposal are to: 1) determine how PAR1 and S1PR1 coordinate endothelial cytoprotective signaling, 2) identify the mechanism(s) by which APC/PAR1 integrates with S1P/S1PR1 to promote cytoprotective signaling, and 3) investigate how PAR1, S1PR1 and -arrestin-2 regulate endothelial barrier integrity in vivo.